Age-related DNA methylation analysis for forensic age estimation using post-mortem blood samples from Japanese individuals.

As one of external visible characteristics (EVCs) in forensic phenotyping, age estimation is essential to providing additional information about a sample donor. With the development of epigenetics, age-related DNA methylation may be used as a reliable predictor of age estimation. With the aim of building a feasible age estimation model for Japanese individuals, 53 CpG sites distributed between 11 candidate genes were selected from previous studies. The DNA methylation level of each target CpG site was identified and measured on a massive parallel platform (synthesis by sequencing, Illumina, California, United States) from 60 forensic blood samples during the initial training phase. Multiple linear regression and quantile regression analyses were later performed to build linear and quantile age estimation models, respectively. Four CpG sites on four genes- ASPA, ELOVL2, ITGA2B, and PDE4C -, were found to be highly correlated with chronological age in DNA samples from Japanese individuals (|R| > 0.75). Subsequently, an independent validation dataset (n = 30) was used to verify and evaluate the performance of the two models. Comparison of mean absolute deviation (MAD) with other indicators showed that both models provide accurate age predictions (MAD: linear = 6.493 years; quantile = 6.243 years). The quantile model, however, can provide the changeable prediction intervals that grow wider with increasing age, and this tendency is consistent with the natural aging process in humans. Hence, the quantile model is recommended in this study.

A stroke patient with impairment of auditory sensory (echoic) memory.

A 42-year-old man suffered damage to the left supra-sylvian areas due to a stroke and presented with verbal short-term memory (STM) deficits. He occasionally could not recall even a single syllable that he had heard one second before. A study of mismatch negativity using magnetoencephalography suggested that the duration of auditory sensory (echoic) memory traces was reduced on the affected side of the brain. His maximum digit span was four with auditory presentation (equivalent to the 1st percentile for normal subjects), whereas it was up to six with visual presentation (almost within the normal range). He simply showed partial recall in the digit span task, and there was no self correction or incorrect reproduction. From these findings, reduced echoic memory was thought to have affected his verbal short-term retention. Thus, the impairment of verbal short-term memory observed in this patient was "pure auditory" unlike previously reported patients with deficits of the phonological short-term store (STS), which is the next higher-order memory system. We report this case to present physiological and behavioral data suggesting impaired short-term storage of verbal information, and to demonstrate the influence of deterioration of echoic memory on verbal STM.

Phenotypic spectrum of patients with PLA2G6 mutation and PARK14-linked parkinsonism.

BACKGROUND: PLA2G6 is the causative gene for infantile neuroaxonal dystrophy, neurodegeneration associated with brain iron accumulation, and Karak syndrome. Based on previous reports, patients with PLA2G6 mutations could show axonal dystrophy, dystonia, dementia, and cerebellar signs. Recently, PLA2G6 was also reported as the causative gene for early-onset PARK14-linked dystonia-parkinsonism. METHODS: To clarify the role of PLA2G6 mutation in parkinsonism, we conducted mutation analysis in 29 selected patients with very early-onset (≤ 30, mean 21.2 ± 8.4 years, ± SD) parkinsonism. These patients had other clinical features (e.g., mental retardation/dementia [14/29], psychosis [15/29], dystonia [11/29], and hyperreflexia [11/29]). RESULTS: Two novel compound heterozygous PLA2G6 mutations were detected (patient A: p.F72L/p.R635Q; patients B1 and B2: p.Q452X/p.R635Q). All 3 patients had early-onset l-dopa-responsive parkinsonism with dementia and frontotemporal lobar atrophy. Disease progression was relatively rapid. SPECT in patient B1 showed frontotemporal lobar hypoperfusion. MRI in patient A showed iron accumulation in the substantia nigra and striatum. CONCLUSIONS: Although the clinical presentation of PLA2G6-associated neurodegeneration was reported to be homogeneous, our findings suggest patients with PLA2G6 mutation could show heterogeneous phenotype such as dystonia-parkinsonism, dementia, frontotemporal atrophy/hypoperfusion, with or without brain iron accumulation. Based on the clinical heterogeneity, the functional roles of PLA2G6 and the roles of PLA2G6 variants including single heterozygous mutations should be further elucidated in patients with atypical parkinsonism, dementia, or Parkinson disease. PLA2G6 mutations should be considered in patients with early-onset l-dopa-responsive parkinsonism and dementia with frontotemporal lobar atrophy.

Significance of levels of IL-6 and IL-8 after trauma: a study of 11 cytokines post-mortem using multiplex immunoassay.

PURPOSE: To identify cytokines useful for diagnosis of traumatic death. METHODS: Post-mortem serum levels of 11 cytokines were assayed for 43 people who died of traumatic injury or from non-traumatic causes. Levels of granulocyte-macrophage colony stimulating factor, gamma interferon, interleukin IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, and tumour necrosis factor-alpha were measured using multiplex immunoassay. RESULTS: Levels of granulocyte-macrophage colony stimulating factor (p<0.01), IL-6 (p<0.001), and IL-8 (p<0.01) among the traumatic group were significantly higher than those among the non-traumatic group. Anatomical trauma severity was also estimated using the total abbreviated injury scale and injury severity score, revealing significant positive correlations between the former and IL-6 (rs=0.6523, p<0.01) and IL-8 levels (rs=0.6584, p<0.01). CONCLUSIONS: Levels of IL-6 and IL-8 assist differentiation between traumatic and non-traumatic death, are useful objective indices of trauma severity and can support a diagnosis of traumatic death.

Polymorphism of 17 STRs by multiplex analysis in Japanese population.

Genotype and distribution of allele frequencies at 17 STRs were studied in 526 unrelated Japanese individuals using the PowerPlex 16 system and the AmpFlSTR Identifiler.

Acral angiokeratoma-like pseudolymphoma: one adolescent and two adults.

In 1988, Ramsay et al proposed an entity of acral pseudolymphomatous angiokeratoma of children (with an abbreviation of APACHE) for the unilateral multiple angiomatous papules affecting the acral region of the extremities of children. We report here similar lesions that developed in the acral portions of 1 female adolescent and 2 women. Histopathologically, they showed pseudolymphomatous features rather than those of angiokeratoma. Thus, the term should be acral angiokeratoma-like pseudolymphoma would be more appropriate than APACHE originally proposed.

Forensic significance of concentrations of ethanol in brain tissues following induced acute subdural hemorrhage.

The concentrations of ethanol in peripheral blood, subdural hematomas and various regions of the brain were determined 1, 2, 4, and 6 h after the induction of a hemorrhage into a subdural space in the right temple of rabbits. The concentrations were compared to cases of intravenous administration of ethanol-free i.v. fluid. Concentrations of ethanol in the subdural hematomas gradually decreased to correspond to those in the peripheral blood. The influence of an intravenous infusion of ethanol-free i.v. fluid was not observed. Concentrations in the brain of the right temporal, parietal and frontal lobes were high and those in the right temporal lobe were maintained during the 6 h of our experiment. Therefore, to determine if a human victim was under the influence of ethanol at the time of injury, we recommend that brain concentrations of ethanol be determined. This is apparently the first study to confirm that the estimation of ethanol in the brain provides a more accurate determination of how much ethanol had been ingested.

Diatom numbers around the continental shelf break.

Diatom concentrations in seawater were examined monthly at four aquatic areas. Diatom concentrations inside a bay showed a monthly variation, but these were detectable. On the other hand, in the open sea around the continental shelf break, there were few diatoms in any season. When a person drowns in the open sea, the diatom test cannot be expected to function reliably.

Acute hemorrhagic edema in a four-year-old Japanese boy.

A case of acute hemorrhagic edema (AHE), a unique form of leukocytoclastic vasculitis limited only to the skin, was seen in a four-year-old boy. It was characterized by an abrupt onset of fever, peripheral edema, and rosette-shaped purpuric plaques. Although the skin lesion might be confused clinically with other dermatoses such as Henoch-Schönlein purpura, Sweet's disease and erythema multiforme, his laboratory data were normal, and the disorder follows a benign course with spontaneous resolution within one week.

Analysis of alpha-synuclein, parkin, tau, and UCH-L1 in a Japanese family with autosomal dominant parkinsonism.

We examined whether autosomal dominant parkinsonism of a Japanese family, Sagamihara family, was due to the mutations of alpha-synuclein, parkin, tau, and UCH-L1, which have been reported as the causal genes for parkinsonism in other families. Restriction-enzyme digestion of polymerase-chain reaction (PCR) amplified genomic DNA fragments of alpha-synuclein exons 3 and 4 detected no point mutation. PCR-amplification of parkin exons 3, 4, 5, 6 and 7 detected no exon deletion. Direct sequencing of PCR-amplified DNA fragments of tau exons 9, 10, 12, and 13 and intron 10, and of UCH-L1 exon 4 revealed that all these exons and intron were normal including a polymorphic nucleotide substitution. These results indicated that the parkinsonism of the Sagamihara family seems not to be due to previously identified point mutations of alpha-synuclein, tau, or UCH-L1, or to exon deletion of parkin.

Correlation between serum IL-6 levels and death: usefulness in diagnosis of "traumatic shock"?

Interleukin-6 (IL-6) has been considered as an important mediator of inflammation. Clinically it is a well-known marker of the severity of injury following major trauma. In this study, the levels of IL-6 in body serum were applied to a traumatic death index. Of ninety victims 55 were men and 35 women, with a mean age of 53.4+/- 19 (S.D.) years. The cases were classified as traumatic deaths (38 cases), non-traumatic deaths other than natural causes of deaths (36 cases), and deaths due to natural causes (16 cases). All samples were collected within 2 days after death. The mean values of IL-6 levels of the traumatic, non-traumatic and disease groups were 8608.97, 2205.65, and 3266.64 pg/ml, respectively. Some cases in non-traumatic and disease cases were beyond 10 000 pg/ml, however, the mean value of the traumatic group was statistically higher than that of the other two groups. Even though several cases had high levels of IL-6 in spite of instantaneous death, the results showed that IL-6 levels are helpful in the diagnosis of traumatic shock.

Occurrence of neutrophils and activated Th1 cells in UVB-induced erythema.

We investigated the sequential changes in infiltrating inflammatory cells and several cytokine levels over a period of 48 h in human back skin exposed to 3 minimal erythematous doses of UVB. The measurement of blood flow, using a laser Doppler method, indicated that UVB-induced erythema reached a peak 12-24 h after irradiation. Immunohistochemically, an increase in the number of CD4+ T cells was observed in perivascular areas 6 h after the UVB treatment and continued for up to 48 h. CD8+ T cells were scarce until 24 h, but their numbers gradually increased thereafter. HLA-DR+ cells were detected perivascularly and interstitially in parallel with the pattern of CD4+ T-cell infiltration. In contrast, neutrophils were found 3 h after UVB exposure and reached a peak at 24 h. Using a RT-PCR method, we demonstrated that mRNAs for the Th1 cytokines (interferon-gamma and interleukin-2), together with a proinflammatory cytokine (interleukin-8), became detectable at 6 h, whereas mRNA for the Th2 cytokine (interleukin-4) was not found at all during the first 48 h. In contrast, we found an increase in mRNA levels for C3 and tumor necrosis factor-alpha even at 3 h, suggesting a relationship between complement activation and accumulating neutrophils. Our results suggest that neutrophils and CD4+ T cells in UVB-induced inflammation play different roles: neutrophils are more closely related to UVB-induced erythema, while T cells appear to be involved in subsequent dermal and epidermal inflammation accompanied by epidermal hyperproliferation.

Regional differences in homicide patterns in five areas of Japan.

This article describes regional differences in the homicide patterns which occurred in Sapporo City and the surrounding area, and in Akita, Ibaraki, Chiba and Toyama prefectures in Japan. Information collected from each case of homicide included factors such as age, sex of the victim and assailant, causes of death, disposition of the offender, relationship between assailant and victim, reasons for criminal action, et al. The statistical features of homicidal episodes among the five different regions showed considerable variation, as follows. The mean death rates for homicide (number of victims per 100,000 of population) during the period 1986-1995 were 0.44 (Sapporo), 0.8 (Akita), 0.58 (Toyama), 0.7 (Ibaraki) and 0.75 (Chiba), respectively. Close family relationship between the victim and assailant was observed in the homicidal acts which occurred in Sapporo, Akita and Toyama. Assailant's relationship to victim was commonly extra-familial in Ibaraki and Chiba-neighboring megalopolis Tokyo, where some events of murder by a foreigner occurred. Homicide by female assailant, murder by mentally abnormal killers and homicide-suicide events were closely associated with family members. And these factors contributed to the considerable number of victims in Sapporo, Akita and Toyama. But, this close family relationship of the victim to the assailant did not correspond with the elevation in the number of deaths, and it was rather inversely related to the higher death rates recognized in Ibaraki and Chiba. This comparative study suggested that rapid urbanization considerably affects regional differences in homicide patterns.

Intracerebral haematoma: traumatic or non-traumatic.

A 35-year-old woman, a chronic alcoholic, died from an intracerebral haematoma 10 hr after she fell downstairs. Some subcutaneous bleeding was seen on the head and face, but there were no new skull fractures and surface contusions of the brain. She appeared to have few predisposing conditions for non-traumatic cerebral haemorrhage. In addition, the haematoma was mainly located "lateral" to the basal ganglia, not where hypertensive bleeding most commonly occurs, and subdural and haemorrhage in the corpus callosum was found with subdural/and subarachnoid haemorrhage. We concluded that on falling a shearing strain from a rotating force produced the intracerebral haemorrhage, but without skull fractures and surface contusions of the brain. She had been admitted to a neurosurgical hospital just 11 months before this incident because of an epidural haemorrhage with left temporal bone fracture. Mild thrombocytopenia was found during that hospitalization. In this report, this abnormality was thought to have some relation to the formation of the huge haematoma occurring after the intracerebral bleeding started.

Distribution of Aconitum alkaloids in body fluids and tissues in a suicidal case of aconite ingestion.

A case involving a suicidal ingestion of Aconitum tubers is presented. A 40-year-old woman in Hokkaido, Japan ingested ground aconite and died of aconite intoxication about 4 h after ingestion. The Aconitum alkaloids were quantitated using gas chromatography-selected ion monitoring from extracts of the body fluids and organs. The blood and urine concentrations of jesaconitine, the main alkaloid of the aconite in this case, were 69.1 ng/mL and 237.8 ng/mL, respectively. Higher values of the alkaloid were demonstrated in the kidneys, the liver, and in the bile rather than other organs or serum, suggesting the alkaloids were eliminated by the liver and kidneys. In the gastrointestinal tract, the highest value of jesaconitine (471.3 ng/g) was in the ileal contents. These findings show that Aconitum alkaloids were found in the liver and kidneys in much higher concentrations than in serum and suggest that they were eliminated not only via urine but also in feces. Feces may be useful to detect Aconitum alkaloid if other biological samples are not available.

Dental identification using digital images via computer network.

Dental identification is a useful scientific method. In Japan, however, there are only a few forensic odontologists; moreover, until now, forensic dental services have only been offered by general dentists. These dentists may not be able to offer such forensic services during office time. For a quick comparison, the authors tried sending digital photos, taken with a 2-million-pixel digital camera, to dental offices via the Internet. If a dental office has Internet access, it is possible for dental charting to be sent directly to the autopsy room. Of course, digital images only provide the first outline. However, when antemortem dental records of the person in question are available at autopsy, a quick comparison can be made.

Ultraviolet B radiation exerts enhancing effects on the production of a complement component, C3, by interferon-gamma-stimulated cultured human epidermal keratinocytes, in contrast to photochemotherapy and ultraviolet A radiation that show suppressive effects.

The present study was designed to investigate by using enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction methods whether photochemotherapy (PUVA) or ultraviolet (UV) B treatment affects C3 production by interferon (IFN)-gamma-stimulated keratinocytes cultured in serum-free medium. The results showed that PUVA and UVA reduced C3 production by IFN-gamma-stimulated epidermal keratinocytes dose-dependently, although the effect of PUVA was stronger than that of UVA alone. Interestingly, UVB induced an enhancement of C3 production at doses ranging from 10 to 50 mJ cm-2. This phenomenon was found at both the protein and mRNA levels. In every experiment, changes in C3 mRNA levels preceded those in its protein levels. Reduced C3 production at higher doses of 75 and 100 mJ cm-2 were probably due to cytotoxic effects of UVB. In our experimental system, PUVA, UVA or UVB treatment did not affect C3 production without IFN-gamma stimulation. Our results suggest that a reduction in C3 production by PUVA treatment may in part explain the efficacy of PUVA in the treatment of inflammatory dermatoses such as psoriasis, while the results of the UVB experiments may partially explain the proinflammatory nature of UVB.

A fatal case of infantile scurvy.

We report a case of infant death due to scurvy, which is very rare in Japan. We initially had little knowledge of the disease and suspected that the bleeding in the body was caused by domestic violence. The case did not fall under the category of the battered child syndrome but the death was caused by ignorance with respect to child care. In addition the parents usually locked the child alone in a room during the day and this is probably a case of neglect.